Project Summary: The intertwined obesity and type 2 diabetes mellitus (T2D) epidemics have focused attention on pathological changes in adipose tissue. However, obesity represents just one of several adiposity- related phenotypes linked to T2D. Visceral fat storage independent of total fat mass is a predictor of T2D. Moreover, there is an overlap in the underlying genetic architecture of T2D and adiposity phenotypes, suggesting shared developmental pathways. In a recent GWAS meta-analysis, we discovered several novel candidate genes linked to deleterious ectopic fat deposition, including the E2 ubiquitin ligase UBE2E2, a lead candidate identified by its genomic proximity to non-coding SNPs associated with visceral fat. UBE2E2 has also been identified by GWAS of T2D. This combined association with a metabolically deleterious body fat distribution and T2D provided rationale to prioritize UBE2E2 for additional functional studies. We have now demonstrated that excision of 100bp regions of non-coding DNA, inclusive of lead UBE2E2-associated SNPs, attenuates expression of UBE2E2, that UBE2E2 loss-of-function in ex vivo adipogenesis assays dramatically inhibits adipocyte differentiation, and that impaired glucose homeostasis arises in a mouse model of UBE2E2 loss-of-function. These preliminary data inform our central hypothesis: that genetic variation in non-coding regions in the UBE2E2 locus results in partial UBE2E2 loss-of-function, impaired adipocyte development as manifested by an obligate shift in favor of ectopic fat deposition, and predisposition to T2DM. We propose to dissect the molecular and biochemical mechanisms underlying the GWAS signals with two interrelated specific aims. In Aim 1, we will interrogate the UBE2E2 locus with Clustered regularly interspaced short palindromic repeats (Crispr) genome editing. We will edit lead SNPs into human adipose derived stem cells and quantify UBE2E2 expression and adipocyte differentiation. With a complementary approach, we will scan the UBE2E2 locus with a Crispr screen to identify putative regulatory regions and their proximity to disease-related SNPs. We will also perform structure-function studies, mutating critical UBE2E2 functional domains, to characterize the biochemical mechanism by which UBE2E2 regulates adipocyte development. Then in Aim 2, we will utilize a murine model of UBE2E2 loss of function to test whether the human traits ? visceral adiposity and T2D ? are recapitulated and moreover whether these phenotypes are attributable to a defect in adipocyte development. Our laboratory has developed methods of precisely quantifying adipogenesis, in vivo, utilizing stable isotope tracers and state-of-the-art mass spectrometric imaging, which we will utilize to quantify adipogenesis. We will couple our quantification of adipogenesis with rigorous characterization of depot specific adiposity and systemic metabolic profiling. Now that GWAS have identified large numbers of candidate genes, their functional characterization is a major bottleneck. This project will establish a pipeline that can be broadly applied to functional validation of variants related to fat phenotypes and cardiometabolic disease susceptibility.